CN110656074B

CN110656074B - Recombinant bacterium for synthesizing hypoxanthine and construction method and application thereof

Info

Publication number: CN110656074B
Application number: CN201810687117.XA
Authority: CN
Inventors: 赵广; 刘敏; 咸漠; 高文杰
Original assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2018-06-28
Filing date: 2018-06-28
Publication date: 2022-06-28
Anticipated expiration: 2038-06-28
Also published as: CN110656074A

Abstract

The invention discloses a recombinant bacterium for synthesizing hypoxanthine as well as a construction method and application thereof, belonging to the technical field of genetic engineering. The recombinant strain takes escherichia coli as an original strain, a transcription repressor gene purR, a phosphogluconate dehydratase gene edd, an adenylate succinate synthetase gene purA and a inosinate dehydrogenase gene guaB on an escherichia coli genome are respectively knocked out, and a PRPP synthetase gene prs with a D128A mutation and a PRPP transamidase gene purF with a K326Q and a P410W double mutation are overexpressed. Meanwhile, the invention also provides a preparation method of the recombinant bacterium and a method for producing hypoxanthine by using the recombinant bacterium. The invention realizes the high-efficiency biosynthesis of hypoxanthine in engineering escherichia coli for the first time. The recombinant strain is suitable for fermentation production of hypoxanthine.

Description

Recombinant bacterium for synthesizing hypoxanthine and construction method and application thereof

Technical Field

The invention relates to a recombinant bacterium for synthesizing hypoxanthine and a construction method and application thereof, belonging to the technical field of genetic engineering.

Background

Purines are important bioactive substances and are involved in the synthesis of genetic information substances in cells. The intermediate metabolites in purine biosynthesis play important roles in aspects of genetic material transmission, signal transport, energy metabolism and the like in cell life processes. In addition, purine and derivatives thereof have wide applications in the medical field, such as diagnosis and treatment of diseases and research fields of life science, etc., and further great interest in synthetic purine and derivatives is brought about. At present, purine-based derivatives have been widely used in pharmaceutical research, for example, purine compounds can induce interferon release, can be used as ligands of corticotropin receptors, or adenosine receptor agonist antagonists, and can exert biological functions by inhibiting various functional enzymes. Purine compounds have a long history in research of antitumor drugs, for example, early mercaptopurine drugs, and later, purine nucleoside drugs such as nelarabine, fludarabine and clorfarabine are found to be used for treating cancers.

At present, purine compound production strains are mainly obtained by using a mutagenesis screening technology, but the production strains usually consume a long time and have low efficiency, and the obtained strains have the defects of instability. In recent years, researchers have attracted attention to synthetic strains of purine compounds obtained by metabolic engineering techniques, as a result of studies on metabolic regulation mechanisms and physiological and biochemical methods of strains. Although the biosynthesis route of purine in cells has been proved, the synthesis route is long and is tightly regulated by multiple layers and different levels in cells, and accumulation of purine is difficult to realize in a natural state. At present, researchers only realize micromolar level synthesis of hypoxanthine in corynebacteria (Corynebacterium glutamicum), and are difficult to meet the requirement of large-scale biosynthesis of purine compounds. Escherichia coli is one of the preferred hosts in the field of microbial synthesis due to low culture cost, high growth speed and simple and convenient gene operation, but no reports of preparing hypoxanthine by using engineering Escherichia coli are available at present. Therefore, the engineering escherichia coli for efficiently synthesizing purine is established by utilizing metabolic engineering technology through regulating purine synthesis pathways, and has important scientific research and application values.

Disclosure of Invention

In order to realize the large-scale synthesis of hypoxanthine by a biological method, the invention provides engineering escherichia coli for synthesizing hypoxanthine, a preparation method of a strain and a method for producing hypoxanthine by using the engineering escherichia coli, and the technical scheme adopted is as follows:

the invention aims to provide a recombinant bacterium for synthesizing hypoxanthine, which takes escherichia coli as an original strain, respectively knocks out a transcription repressor gene purR, a phosphogluconate dehydratase gene edd, an adenylate succinate synthetase gene purA and a hypoxanthine dehydrogenase gene guaB on an escherichia coli genome, and overexpresses a PRPP synthetase gene prs with a D128A mutation, a PRPP transamidase gene purF with a K326Q mutation and a P410W double mutation.

Preferably, the Gene ID of the transcriptional repressor Gene purR is 945226; the Gene ID of the phosphogluconate dehydratase Gene edd is 946362; the Gene ID of the adenylosuccinate synthetase Gene purA is 948695; the Gene ID of the inosinic acid dehydrogenase Gene guaB is 946985; the nucleotide sequence of the PRPP synthetase gene prs with the D128A mutation is shown as SEQ ID NO. 1; the nucleotide sequence of the K326Q and P410W double-mutation PRPP amidotransferase gene purF is shown as SEQ ID NO. 2.

Preferably, Escherichia coli W3110 is used as the starting strain.

The invention also provides a construction method of any one of the recombinant bacteria, which comprises the following steps:

1) respectively knocking out a transcription repressor gene purR, a phosphogluconate dehydratase gene edd, an adenylate succinate synthetase gene purA and an inosinate dehydrogenase gene guaB of a genome of escherichia coli W3110 by taking escherichia coli W3110 as an initial strain to obtain a mutant strain W3110 delta purR delta edd delta purA delta guaB; the Gene ID of the transcriptional repressor Gene purR is 945226; the Gene ID of the phosphogluconate dehydratase Gene edd is 946362; the Gene ID of the adenylosuccinate synthetase Gene purA is 948695; the Gene ID of the inosinic acid dehydrogenase Gene guaB is 946985;

2) cloning to obtain a PRPP synthetase gene prs with a D128A mutation (D128A) and PRPP transamidase purF with K326Q and P410W double mutations (K326Q and P410W) by a fragment bridging method; the nucleotide sequence of the D128A mutant PRPP synthetase gene prs is shown as SEQ ID NO. 1; the nucleotide sequence of the K326Q and P410W double-mutation PRPP amidotransferase gene purF is shown as SEQ ID NO. 2;

3) connecting the PRPP transamidase gene purF (K326Q, P410W) obtained in the step 2) to a plasmid vector to obtain a recombinant plasmid I;

4) Connecting the PRPP synthetase gene prs (D128A) obtained in the step 2) to a recombinant plasmid I to obtain a recombinant plasmid II;

5) introducing the recombinant plasmid II obtained in the step 4) into the mutant strain obtained in the step 1) to obtain a recombinant strain.

Preferably, the plasmid vector of step 3) is plasmid pACYCDuet-1.

Preferably, the method comprises the steps of:

1) knocking out a transcription repressor gene purR, a phosphogluconate dehydratase gene edd, an adenylate succinate synthetase gene purA and an inosinate dehydrogenase gene guaB of an escherichia coli W3110 genome respectively by using a P1 phage transduction method to obtain a mutant strain W3110 delta purR delta edd delta purA delta guaB; the Gene ID of the transcription repressor Gene purR is 945226; the Gene ID of the phosphogluconate dehydratase Gene edd is 946362; the Gene ID of the adenylosuccinate synthetase Gene purA is 948695; the Gene ID of the inosinic acid dehydrogenase Gene guaB is 946985;

2) taking genome DNA of escherichia coli as a template, cloning an upstream fragment of a K326Q mutant PRPP amidotransferase gene purF by using primers SEQ ID No.4 and SEQ ID No.6, cloning a downstream fragment of a K326Q mutant PRPP amidotransferase gene purF by using primers SEQ ID No.5 and SEQ ID No.7, and bridging the upstream and downstream fragments of the K326Q mutant PRPP amidotransferase gene purF by using a fragment bridging method by using primers SEQ ID No.4 and SEQ ID No.5 to obtain a K326Q mutant PRPP amidotransferase gene purF shown in SEQ ID No. 3; taking the PRPP amidotransferase gene purF with K326Q mutation as a template, cloning an upstream fragment of the PRPP amidotransferase gene purF with P410W mutation by using primers SEQ ID NO.4 and SEQ ID NO.8, cloning a downstream fragment of the PRPP amidotransferase gene purF with P410W mutation by using primers SEQ ID NO.5 and SEQ ID NO.9, bridging the upstream and downstream fragments of the PRPP amidotransferase gene purF with P410W mutation by using a fragment bridging method by using primers SEQ ID NO.4 and SEQ ID NO.5, and obtaining the PRPP amidotransferase gene purF with K326Q and P410W double mutations shown in SEQ ID NO. 2;

3) Connecting the K326Q and P410W double-mutation PRPP transamidase gene purF (K326Q and P410W) obtained in the step 2) to a plasmid vector pACYCDuet-1 to obtain a recombinant plasmid pACYCDuet1-purF (K326Q and P410W);

4) taking genome DNA of escherichia coli as a template, cloning an upstream fragment of a PRPP synthetase gene prs mutated by D128A by using primers SEQ ID NO.10 and SEQ ID NO.11, cloning a downstream fragment of the PRPP synthetase gene prs mutated by D128A by using primers SEQ ID NO.12 and SEQ ID NO.13, and bridging the upstream fragment and the downstream fragment of the PRPP synthetase gene prs mutated by D128A by using primers SEQ ID NO.10 and SEQ ID NO.13 by using a fragment bridging method to obtain the PRPP synthetase gene prs mutated by D128A shown as SEQ ID NO. 1;

5) connecting the PRPP synthetase gene prs (D128A) obtained in the step 4) to the recombinant plasmid pACYCDuet1-purF (K326Q, P410W) obtained in the step 3) to obtain a recombinant plasmid pACYCDuet1-prs (D128A) -purF (K326Q, P410W);

6) the recombinant plasmid pACYCDuet1-prs (D128A) -purF (K326Q, P410W) obtained in the step 5) was introduced into the recipient cell W3110. delta. purR. delta. edd. delta. purA. delta. guaB obtained in the step 1) to obtain a recombinant strain.

The method for introducing the recombinant plasmid into the recipient cell of the present invention may employ a heat shock transformation method.

The invention also provides application of any one of the recombinant bacteria in producing hypoxanthine and producing hypoxanthine derivatives.

Preferably, the application is to activate the recombinant bacteria, and the activated recombinant bacteria are inoculated into LB liquid culture medium containing chloramphenicol for fermentation culture.

More preferably, the fermentation culture is to culture the recombinant bacteria to OD600 of 8-12 under the conditions of culture temperature of 37 ℃, stirring speed of 400-800rpm and pH value of 6.5-7.5, then to add inducer isopropyl thiogalactoside IPTG to the final concentration of 100 mu M, and to use 50-80% by mass of glucose stock solution to continue the fed-batch fermentation for 96h and then to finish the fermentation.

Preferably, the concentration of the chloramphenicol in the LB liquid medium is 50 mg/L.

Preferably, the inoculation is to inoculate the recombinant strain seed liquid into LB liquid culture medium containing chloramphenicol according to the inoculation amount of 2-5% (v/v).

The Gene ID of the transcription repressor protein Gene purR is 945226; the Gene ID of the phosphogluconate dehydratase Gene edd is 946362; the Gene ID of the adenylosuccinate synthetase Gene purA is 948695; the Gene ID of the inosinic acid dehydrogenase Gene guaB is 946985.

In the present invention, PRPP synthetase Gene prs is derived from Escherichia coli (Escherichia coli), and its Gene ID is 945772. The PRPP synthetase gene prs with the D128A mutation, namely the PRPP synthetase gene prs contains the D128A mutation, and the nucleotide sequence of the PRPP synthetase gene prs is shown as SEQ ID NO. 1. D128A indicates that the 128 th amino acid D is mutated to A.

The PRPP amidotransferase Gene purF is derived from Escherichia coli (Escherichia coli), and the Gene ID of the PRPP amidotransferase Gene purF is 946794. The PRPP transamidase gene purF with double mutations of K326Q and P410W, namely the PRPP transamidase gene purF contains double mutations of K326Q and P410W, and the nucleotide sequence of the PRPP transamidase gene purF is shown as SEQ ID No. 2. The double mutation of K326Q and P410W means that the mutation of the amino acid K at the 326 th position is Q, and the mutation of the amino acid P at the 410 th position is W.

The P1 phage transfer method of the present invention is one of the techniques commonly used for gene knock-out in the art, and can be performed according to standard procedures.

The definitions and abbreviations referred to in the present invention are as follows:

transcriptional repressor gene: purR

Phosphogluconate dehydratase gene: edd

Adenylosuccinate synthetase gene: purA

Inosinic acid dehydrogenase gene: guaB

PRPP synthetase gene: prs

PRPP transamidase gene: purF

Coli (Escherichia coli): coli

The term "heat shock transformation" or "heat transformation" as used herein refers to one of the techniques of transfection in molecular biology, which is used to integrate a foreign gene into a host gene and stably express the gene, and utilizes the fact that after heat shock, a cell membrane develops a slit, the foreign gene is introduced into the host gene or a foreign plasmid is introduced into a host protoplast, and then heat shock transformation or heat transformation, etc.

"overexpression" or "overexpression" in the present invention means that a specific gene is expressed in an organism in a large amount, which exceeds a normal level (i.e., wild-type expression level), and can be achieved by enhancing endogenous expression or introducing a foreign gene.

The application of the recombinant bacterium in fermentation production of hypoxanthine and derivative products thereof is within the protection scope of the invention.

The invention has the beneficial effects that:

the invention modifies the biosynthesis pathway of hypoxanthine, constructs an engineering strain capable of realizing large-scale accumulation of hypoxanthine, and specifically knocks out transcription repressor (PurR), phosphogluconate dehydratase (Edd), adenylate succinate synthetase (PurA), inosinate dehydrogenase (GuaB) on a genome in escherichia coli, and over-expresses PRPP synthetase (Prs) with D128A mutation and PRPP transamidase (PurF) with K326Q and P410W double mutations.

The invention takes the mode strain of the escherichia coli as a host bacterium, establishes a recombinant bacterium for producing the hypoxanthine through metabolic engineering technology by regulating the biosynthesis way of the purine, establishes the high-efficiency biosynthesis technology for preparing the hypoxanthine by the engineering escherichia coli for the first time, and makes up the blank of preparing the hypoxanthine by the engineering escherichia coli. Provides a new technical method for the high-efficiency biosynthesis of hypoxanthine and derivatives thereof.

In the prior art, the hypoxanthine is only synthesized in a micromole level in corynebacteria, the synthesis of the hypoxanthine in escherichia coli is not reported, and the recombinant escherichia coli constructed by the invention can realize the accumulation of the hypoxanthine to 796mg/L (converted to 5.85 mM/L).

Drawings

FIG. 1 is a plasmid map of recombinant plasmid pACYCDuet1-prs (D128A) -purF (K326Q, P410W).

FIG. 2 shows the shake flask fermentation detection result of the recombinant bacterium Q2955;

(A is the LC-MS detection result of the Q2955 fermentation product, and B is the secondary mass spectrum of hypoxanthine).

FIG. 3 shows the results of 5L-fermentor detection of recombinant bacterium Q2955.

FIG. 4 is a diagram showing gene knockout by the P1 phage transfer method.

Detailed Description

The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.

The materials, reagents, apparatus and methods used in the following examples, which are not specifically illustrated, are all conventional in the art and are commercially available.

The enzyme reagent is purchased from MBI Fermentas company, the kit for extracting plasmid and the kit for recovering DNA fragment are purchased from American OMEGA company, and the corresponding operation steps are carried out according to the product instruction; all media were formulated with deionized water unless otherwise indicated.

The formula of the culture medium is as follows:

1) LB culture medium: 5g/L yeast powder, 10g/L NaCl, 10g/L peptone and the balance water at 121 ℃,

sterilizing for 20 min.

2) Fermentation production culture medium

Fermentation medium: 5g/L yeast powder, 10g/L NaCl, 10g/L peptone and 20g/L glucose.

During the actual culture process, antibiotics can be added to the culture medium at a certain concentration to maintain the stability of plasmids, such as 50mg/L chloramphenicol.

The nucleotide sequences of the primers involved in the following experimental procedures are shown in Table 1.

TABLE 1 nucleotide sequences of primers used in the construction of recombinant bacteria of the invention

Example 1: construction of recombinant bacterium

One, gene knockout (P1 phage transfer method, principle is shown in FIG. 4)

The knockout of the transcriptional repressor gene purR is carried out according to the following method:

knocking out a transcription repressor Gene purR (Gene ID:945226) of an Escherichia coli W3110 genome, namely, infecting a donor strain JW1650 of a Keio Collection library by using P1 phage by using a P1 phage transfer method, preparing a donor strain cracking library, transducing an acceptor strain Escherichia coli W3110 by using the cracking library of the JW1650, and obtaining a mutant strain W3110 delta purR of which the transcription repressor Gene purR is knocked out;

1) phage activation

10ml of sterile EP tube was added with 4ml of 0.4% agar medium melted by heating, 400. mu.L of overnight cultured donor strain JW1650 (the donor strain is derived from a Keio Collection library and commercially available, and the Keio Collection library was constructed by the method described in "Baba T, et al.Construction of Escherichia coli K-12in-frame, single-gene knock-out variants: the Keio Collection. molecular Systems Biology 2006,2(1): 1-11"), 10. mu.L of stock phage solution was added, the solution was mixed and poured onto LB no-antibody plate, and cultured in a wet environment at 37 ℃ until plaques appeared.

2) Collection of phage lytic libraries

Scraping all the semisolid culture medium on the plate to a 10ml sterile EP tube, adding 3ml LB liquid culture medium, adding 400 mu L chloroform, shaking, centrifuging, collecting the supernatant to another sterile EP tube, and adding 400 mu L chloroform, wherein the solution is the JW1650 donor bacterium lysis library.

3) Transduction of

Overnight culturing recipient bacterium E.coli W3110, mixing the JW1650 donor bacterium lysis library prepared in the step 2) with the recipient bacterium E.coli W3110 in different concentrations, transducing, coating a resistant plate, overnight culturing until a single clone grows out, and verifying positive cloning by using primers SEQ ID No.14 and SEQ ID No.15 to obtain a mutant strain W3110 delta purR with the transcription repressor gene purR knocked out.

The phosphogluconate dehydratase gene edd is knocked out according to the following method:

the phosphogluconate dehydratase Gene edd (Gene ID:946362) of the E.coli W3110 genome was knocked out by the P1 phage transfer method. The operation process of knocking out edd gene by P1 phage transduction refers to the operation process of knocking out transcriptional repressor gene purR. The difference is that: step 1) the donor strain activated by the phage is JW1840 derived from a Keio Collection library, step 2) the JW1840 donor strain lysis library is obtained by Collection, step 3) transduction is carried out after the JW1840 donor strain lysis library prepared in the step 2) is mixed with the recipient strain W3110 delta purR at different concentrations, verification of positive cloning is carried out by primers SEQ ID No.14 and SEQ ID No.16, and the mutant strain W3110 delta purR delta edd with the transcription repressor gene purR and the phosphogluconate dehydratase gene edd knocked out is obtained.

The adenylosuccinate synthetase gene purA is knocked out according to the following method:

the adenylosuccinate synthetase Gene purA (Gene ID:948695) of the E.coli W3110 genome was knocked out by the P1 phage transfer method. The operation process of knocking out purA gene by the P1 phage transduction method refers to the operation process of knocking out transcription repressor gene purR. The difference is that: step 1) the donor strain with activated phage is JW4135 from Keio Collection library, step 2) the obtained JW4135 donor strain lysis library is collected, step 3) transduction is to mix the JW4135 donor strain lysis library prepared in step 2) with the acceptor strain W3110 delta purR delta edd at different concentrations and conduct transduction, verification of positive cloning is conducted through primers SEQ ID No.14 and SEQ ID No.17, and the mutant strain W3110 delta purR delta purA with the transcription repressor gene purR, the phosphogluconate dehydratase gene edd and the adenylate succinate synthetase gene purA knocked out is obtained.

The knockout of inosinic acid dehydrogenase gene guaB was carried out as follows:

the inosinic acid dehydrogenase Gene guaB (Gene ID:946985) of the E.coli W3110 genome was knocked out by P1 phage transfer method. The P1 phage transduction knockout of guaB gene is performed according to the knockout procedure of the transcriptional repressor gene purR. The difference is as follows: step 1) the donor strain activated by the phage is JW5401 from a Keio Collection library, step 2) the JW5401 donor strain lysis library is obtained by Collection, step 3) transduction is to mix the JW5401 donor strain lysis library prepared in the step 2) with the recipient strain W3110 delta purR delta edd delta purA at different concentrations and then carry out transduction, and primers SEQ ID NO.14 and SEQ ID NO.18 are used for verifying positive cloning, so that the mutant strain W3110 delta purR delta edd delta purA delta guaB with the transcription repressor gene purR, the phosphogluconate dehydratase gene edd, the adenylate succinate synthetase gene purA and the inosinate dehydrogenase gene guaB knocked out is obtained.

Secondly, the construction process of the recombinant plasmid pACYCDuet1-prs (D128A) -purF (K326Q, P410W)

1) Taking genome DNA of escherichia coli as a template, cloning an upstream fragment of a K326Q mutant PRPP amidotransferase gene purF by using primers SEQ ID No.4 and SEQ ID No.6, cloning a downstream fragment of a K326Q mutant PRPP amidotransferase gene purF by using primers SEQ ID No.5 and SEQ ID No.7, and bridging the upstream and downstream fragments of the K326Q mutant PRPP amidotransferase gene purF by using primers SEQ ID No.4 and SEQ ID No.5 by using a fragment bridging method to obtain a K326Q mutant PRPP amidotransferase gene purF shown in SEQ ID No. 3; taking K326Q mutant PRPP amidotransferase gene purF as a template, cloning an upstream fragment of P410W mutant PRPP amidotransferase gene purF by using primers SEQ ID No.4 and SEQ ID No.8, cloning a downstream fragment of P410W mutant PRPP amidotransferase gene purF by using primers SEQ ID No.5 and SEQ ID No.9, and bridging the upstream and downstream fragments of P410W mutant PRPP amidotransferase gene purF by using a fragment bridging method and primers SEQ ID No.4 and SEQ ID No.5 to obtain K326Q and P410W double mutant PRPP amidotransferase gene purF shown in SEQ ID No. 2;

2) Carrying out SacI and HindIIII double enzyme digestion on K326Q and P410W double-mutation PRPP transamidase genes purF (K326Q and P410W) obtained in the step 1) and a vector pACYCDuet-1, recovering target fragments purF (K326Q and P410W) and the vector pACYCDuet-1 after enzyme digestion by using a recovery kit, connecting by using T4DNA ligase, transforming E.coli DH5 alpha by using a connecting product, and screening positive clones to obtain recombinant plasmids pACYCDuet1-purF (K326Q and P410W);

3) taking genome DNA of escherichia coli as a template, cloning an upstream fragment of a PRPP synthetase gene prs mutated by D128A by using primers SEQ ID NO.10 and SEQ ID NO.11, cloning a downstream fragment of a PRPP synthetase gene prs mutated by D128A by using primers SEQ ID NO.12 and SEQ ID NO.13, and bridging the upstream and downstream fragments of the PRPP synthetase gene prs mutated by D128A by using primers SEQ ID NO.10 and SEQ ID NO.13 by using a fragment bridging method to obtain the PRPP synthetase gene prs mutated by D128A shown as SEQ ID NO. 1;

4) carrying out double digestion on the PRPP synthetase gene prs (D128A) with the D128A mutation obtained in the step 3) and the recombinant plasmid pACYCDuet1-purF (K326Q and P410W) obtained in the step 2) through BamHI and SacI, recovering a target fragment prs (D128A) and a vector pACYCDuet1-purF (K326Q and P410W) after digestion by using a recovery kit, connecting by using T4DNA ligase, transforming E.coli DH5 alpha by using a connecting product, and screening positive clones to obtain a recombinant plasmid pACYCDuet1-prs (D128A) -purF (K326Q and P410W);

Third, construction of recombinant strains

Mutant strain W3110. delta. purR. delta. edd. delta. purA. delta. guaB was made competent according to the procedure of TAKARA competent kit, and recombinant plasmid pACYCDuet1-prs (D128A) -purF (K326Q, P410W) was transformed into mutant strain W3110. delta. purR. delta. edd. delta. purA. delta. guaB competent cells by heat shock method to obtain recombinant strain, accession number Q2955.

EXAMPLE 2 Shake flask fermentation test of recombinant strains

In this example, two experiments were performed to illustrate the effect of the present invention, and the specific experimental methods are as follows:

control group: coli W3110, a wild strain,

experimental groups: the recombinant strain Q2955 is used as a recombinant strain,

1) the activated wild strain E.coli W3110 and the recombinant strain Q2955 were inoculated into a 250mL shake flask containing 50mL of a fermentation medium (containing 50mg/L of chloramphenicol) at a ratio of 1:100, and shake-cultured at 37 ℃ and 180 rpm. OD₆₀₀When the concentration reaches about 0.6, adding 100 mu M IPTG to induce expression, and after induction, placing at 30 ℃ and continuing culturing at 180rpm for 96h until the fermentation is finished.

2) Centrifuging 1mL of fermentation liquid at 4 ℃ and 12000rpm for 10min, taking supernatant, filtering with a 0.22-micron filter membrane, and detecting the fermentation product by using LC-MS.

3) LC-MS detection (FIG. 2) demonstrated that no hypoxanthine was detected by the wild strain, whereas the experimental group yielded the product hypoxanthine. At the fermentation level of a 250mL shake flask, the yield of the hypoxanthine of the engineering strain Q2955 is 72mg/L, and the characteristic peak of the secondary mass spectrum is completely consistent with that of the standard product. This indicates that the wild strain cannot naturally accumulate hypoxanthine without metabolic engineering of the hypoxanthine synthesis pathway of the present invention. Therefore, the invention provides a recombinant bacterium for efficiently synthesizing hypoxanthine.

EXAMPLE 3 fermenter experiment with recombinant strains

1) The recombinant strain Q2955 was inoculated into 3mL of liquid LB medium and cultured overnight at 37 ℃ with shaking at 180rpm as a primary seed solution.

2) The activated primary seed solution was inoculated into a 250mL shake flask containing 50mL fermentation medium (containing 50mg/L chloramphenicol) at a ratio of 1:100, and shake-cultured at 37 ℃ and 180rpm for 4 hours to give a secondary seed solution.

3) Inoculating the secondary seed liquid into a fermentation tank according to the inoculation amount of 2% of the volume of the culture medium, wherein the culture temperature is 37 ℃, the stirring speed is 400-800rpm, 20% dissolved oxygen is related to the rotating speed, the pH value is automatically adjusted to about 7.0 by using ammonia water and 10% sulfuric acid, the recombinant cells are cultured until the OD600 is 10, then adding an inducer IPTG to the final concentration of 100 mu M, and continuously feeding and fermenting 96 by using a glucose stock solution with the mass fraction of 60%.

4) Periodically taking 1mL of fermentation liquor in the fermentation process, centrifuging at 4 ℃ and 12000rpm for 10min, taking supernatant, filtering with a 0.22-micron filter membrane, and detecting the fermentation product by using LC-MS.

5) LC-MS detection shows that hypoxanthine is accumulated continuously in the fermentation process (figure 3), and the hypoxanthine is fermented for 96h to reach 796 mg/L.

It will be appreciated by those skilled in the art that each of the above steps is performed according to standard molecular cloning techniques.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> institute for bioenergy and Process in Qingdao of Chinese academy of sciences

<120> recombinant bacterium for synthesizing hypoxanthine and construction method and application thereof

<130> 1

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 948

<212> DNA

<213> D128A mutant PRPP synthetase gene prs

<400> 1

gtgcctgata tgaagctttt tgctggtaac gccaccccgg aactagcaca acgtattgcc 60

aaccgcctgt acacttcact cggcgacgcc gctgtaggtc gctttagcga tggcgaagtc 120

agcgtacaaa ttaatgaaaa tgtacgcggt ggtgatattt tcatcatcca gtccacttgt 180

gcccctacta acgacaacct gatggaatta gtcgttatgg ttgatgccct gcgtcgtgct 240

tccgcaggtc gtatcaccgc tgttatcccc tactttggct atgcgcgcca ggaccgtcgc 300

gtccgttccg ctcgtgtacc aatcactgcg aaagtggttg cagacttcct ctccagcgtc 360

ggtgttgacc gtgtgctgac agtggcgctg cacgctgaac agattcaggg tttcttcgac 420

gttccggttg ataacgtatt tggtagcccg atcctgctgg aagacatgct gcagctgaat 480

ctggataacc caattgtggt ttctccggac atcggcggcg ttgtgcgtgc ccgcgctatc 540

gctaagctgc tgaacgatac cgatatggca atcatcgaca aacgtcgtcc gcgtgcgaac 600

gtttcacagg tgatgcatat catcggtgac gttgcaggtc gtgactgcgt actggtcgat 660

gatatgatcg acactggcgg tacgctgtgt aaagctgctg aagctctgaa agaacgtggt 720

gctaaacgtg tatttgcgta cgcgactcac ccgatcttct ctggcaacgc ggcgaacaac 780

ctgcgtaact ctgtaattga tgaagtcgtt gtctgcgata ccattccgct gagcgatgaa 840

atcaaatcac tgccgaacgt gcgtactctg accctgtcag gtatgctggc cgaagcgatt 900

cgtcgtatca gcaacgaaga atcgatctct gccatgttcg aacactaa 948

<210> 2

<211> 1518

<212> DNA

<213> K326Q, P410W double-mutated PRPP transamidase gene purF

<400> 2

atgtgcggta ttgtcggtat cgccggtgtt atgccggtta accagtcgat ttatgatgcc 60

ttaacggtgc ttcagcatcg cggtcaggat gccgccggca tcatcaccat agatgccaat 120

aactgcttcc gtttgcgtaa agcgaacggg ctggtgagcg atgtatttga agctcgccat 180

atgcagcgtt tgcagggcaa tatgggcatt ggtcatgtgc gttaccccac ggctggcagc 240

tccagcgcct ctgaagcgca gccgttttac gttaactccc cgtatggcat tacgcttgcc 300

cacaacggca atctgaccaa cgctcacgag ttgcgtaaaa aactgtttga agaaaaacgc 360

cgccacatca acaccacttc cgactcggaa attctgctta atatcttcgc cagcgagctg 420

gacaacttcc gccactaccc gctggaagcc gacaatattt tcgctgccat tgctgccaca 480

aaccgcttaa tccgcggcgc gtatgcctgt gtggcgatga ttatcggcca cggtatggtt 540

gctttccgcg atccaaacgg gattcgtccg ctggtactgg gaaaacgtga tattgacgag 600

aaccgtacag aatatatggt cgcttccgaa agcgtagcgc tcgatacgct gggctttgat 660

ttcctgcgtg acgtcgcgcc gggcgaagcg atttacatca ctgaagaagg gcagttgttt 720

acccgtcaat gtgctgacaa tccggtcagc aatccgtgcc tgtttgagta tgtatacttt 780

gcccgcccgg actcgtttat cgacaaaatt tccgtttaca gcgcgcgtgt gaatatgggc 840

acgaaactgg gcgagaaaat tgcccgcgaa tgggaagatc tggatatcga cgtggtgatc 900

ccgatcccag aaacctcgtg tgatatcgcg ctggaaattg ctcgtattct gggcaaaccg 960

taccgccagg gcttcgttca gaaccgctat gttggccgca cctttatcat gccgggccag 1020

cagctgcgtc gtaagtccgt gcgccgtaaa ctgaatgcca accgcgccga gttccgcgat 1080

aaaaacgtcc tgctggtcga cgactccatc gtccgtggca ccacttctga gcagattatc 1140

gagatggcac gcgaagccgg agcgaagaaa gtgtacctcg cttctgcggc accggaaatt 1200

cgcttcccga acgtttatgg tattgatatg tggagcgcca cggaactgat cgctcacggt 1260

cgcgaagttg atgaaattcg ccagatcatc ggtgctgacg ggttgatttt ccaggatctg 1320

aacgatctga tcgacgccgt tcgcgctgaa aatccggata tccagcagtt tgaatgctcg 1380

gtgttcaacg gcgtctacgt caccaaagat gttgatcagg gctacctcga tttcctcgat 1440

acgttacgta atgatgacgc caaagcagtg caacgtcaga acgaagtgga aaatctcgaa 1500

atgcataacg aaggatga 1518

<210> 3

<211> 1518

<212> DNA

<213> K326Q mutant PRPP transamidase gene purF

<400> 3

atgtgcggta ttgtcggtat cgccggtgtt atgccggtta accagtcgat ttatgatgcc 60

ttaacggtgc ttcagcatcg cggtcaggat gccgccggca tcatcaccat agatgccaat 120

aactgcttcc gtttgcgtaa agcgaacggg ctggtgagcg atgtatttga agctcgccat 180

atgcagcgtt tgcagggcaa tatgggcatt ggtcatgtgc gttaccccac ggctggcagc 240

tccagcgcct ctgaagcgca gccgttttac gttaactccc cgtatggcat tacgcttgcc 300

cacaacggca atctgaccaa cgctcacgag ttgcgtaaaa aactgtttga agaaaaacgc 360

cgccacatca acaccacttc cgactcggaa attctgctta atatcttcgc cagcgagctg 420

gacaacttcc gccactaccc gctggaagcc gacaatattt tcgctgccat tgctgccaca 480

aaccgcttaa tccgcggcgc gtatgcctgt gtggcgatga ttatcggcca cggtatggtt 540

gctttccgcg atccaaacgg gattcgtccg ctggtactgg gaaaacgtga tattgacgag 600

aaccgtacag aatatatggt cgcttccgaa agcgtagcgc tcgatacgct gggctttgat 660

ttcctgcgtg acgtcgcgcc gggcgaagcg atttacatca ctgaagaagg gcagttgttt 720

acccgtcaat gtgctgacaa tccggtcagc aatccgtgcc tgtttgagta tgtatacttt 780

gcccgcccgg actcgtttat cgacaaaatt tccgtttaca gcgcgcgtgt gaatatgggc 840

acgaaactgg gcgagaaaat tgcccgcgaa tgggaagatc tggatatcga cgtggtgatc 900

ccgatcccag aaacctcgtg tgatatcgcg ctggaaattg ctcgtattct gggcaaaccg 960

taccgccagg gcttcgttca gaaccgctat gttggccgca cctttatcat gccgggccag 1020

cagctgcgtc gtaagtccgt gcgccgtaaa ctgaatgcca accgcgccga gttccgcgat 1080

aaaaacgtcc tgctggtcga cgactccatc gtccgtggca ccacttctga gcagattatc 1140

gagatggcac gcgaagccgg agcgaagaaa gtgtacctcg cttctgcggc accggaaatt 1200

cgcttcccga acgtttatgg tattgatatg ccgagcgcca cggaactgat cgctcacggt 1260

cgcgaagttg atgaaattcg ccagatcatc ggtgctgacg ggttgatttt ccaggatctg 1320

aacgatctga tcgacgccgt tcgcgctgaa aatccggata tccagcagtt tgaatgctcg 1380

gtgttcaacg gcgtctacgt caccaaagat gttgatcagg gctacctcga tttcctcgat 1440

acgttacgta atgatgacgc caaagcagtg caacgtcaga acgaagtgga aaatctcgaa 1500

atgcataacg aaggatga 1518

<210> 4

<211> 70

<212> DNA

<213> purF-up-5'

<400> 4

ccggagctcc tttacacttt aagcttttta tgtttatgtt gtgtggaatt gagcaaatca 60

cagctgatcc 70

<210> 5

<211> 29

<212> DNA

<213> purF-down-3'

<400> 5

ccgaagcttc gcagaacctg taataagcg 29

<210> 6

<211> 20

<212> DNA

<213> purF-up(K326Q)-3'

<400> 6

aacgaagccc tggcggtacg 20

<210> 7

<211> 42

<212> DNA

<213> purF-down(K326Q)-5'

<400> 7

cgtaccgcca gggcttcgtt cagaaccgct atgttggccg ca 42

<210> 8

<211> 20

<212> DNA

<213> purF-up(P410W)-3'

<400> 8

catatcaata ccataaacgt 20

<210> 9

<211> 43

<212> DNA

<213> purF-down(P410W)-5'

<400> 9

acgtttatgg tattgatatg tggagcgcca cggaactgat cgc 43

<210> 10

<211> 30

<212> DNA

<213> prs(D128A)-up-5'

<400> 10

ccgggatccg ccattgcaca gagccatgct 30

<210> 11

<211> 20

<212> DNA

<213> prs(D128A)-up-3'

<400> 11

ccactgtcag cacacggtca 20

<210> 12

<211> 42

<212> DNA

<213> prs(D128A)-down-5'

<400> 12

tgaccgtgtg ctgacagtgg cgctgcacgc tgaacagatt ca 42

<210> 13

<211> 29

<212> DNA

<213> prs(D128A)-down-3'

<400> 13

ccggagctcc cagcaagcgt cgatcagag 29

<210> 14

<211> 20

<212> DNA

<213> Kan-3'

<400> 14

ggtgagatga caggagatcc 20

<210> 15

<211> 20

<212> DNA

<213> ID-purR-5'

<400> 15

tccacgctta cactatttgc 20

<210> 16

<211> 21

<212> DNA

<213> ID-edd-5'

<400> 16

atgatcttgc gcagattgta g 21

<210> 17

<211> 21

<212> DNA

<213> ID-purA-5'

<400> 17

gtaactctga aaaagcgatg g 21

<210> 18

<211> 20

<212> DNA

<213> ID-guaB-5'

<400> 18

gcaggttatt cagtcgatag 20

Claims

1. The recombinant strain for synthesizing hypoxanthine is characterized in that escherichia coli is used as an original strain, a transcription repressor gene purR, a phosphogluconate dehydratase gene edd, an adenylate succinate synthetase gene purA and a hypoxanthine dehydrogenase gene guaB on an escherichia coli genome are knocked out respectively, and a PRPP synthetase gene prs with a D128A mutation, a PRPP transamidase gene purF with a K326Q mutation and a P410W double mutation are overexpressed.

2. The recombinant bacterium according to claim 1, wherein the Gene ID of the transcriptional repressor Gene purR in NCBI is 945226; the Gene ID of the phosphogluconate dehydratase Gene edd in NCBI is 946362; the Gene ID of the adenylosuccinate synthetase Gene purA in NCBI is 948695; the Gene ID of the inosinic acid dehydrogenase Gene guaB in NCBI is 946985; the nucleotide sequence of the PRPP synthetase gene prs with the D128A mutation is shown as SEQ ID NO. 1; the nucleotide sequence of the K326Q and P410W double-mutation PRPP amidotransferase gene purF is shown as SEQ ID NO. 2.

3. The recombinant bacterium according to claim 1, wherein Escherichia coli W3110 is used as a starting strain.

4. A method for constructing a recombinant bacterium according to any one of claims 1 to 3, comprising the steps of:

1) respectively knocking out a transcription repressor gene purR, a phosphogluconate dehydratase gene edd, an adenylate succinate synthetase gene purA and an inosinate dehydrogenase gene guaB of an escherichia coli W3110 genome by taking escherichia coli W3110 as an original strain to obtain a mutant strain W3110 delta purR delta edd delta purA delta guaB;

2) cloning to obtain D128A mutated PRPP synthetase genes prs, K326Q and P410W double mutated PRPP transamidase purF by utilizing a fragment bridging method; the nucleotide sequence of the PRPP synthetase gene prs with the D128A mutation is shown as SEQ ID NO. 1; the nucleotide sequence of the K326Q and P410W double-mutation PRPP amidotransferase gene purF is shown as SEQ ID NO. 2;

3) Connecting the PRPP transamidase gene purF obtained in the step 2) to a plasmid vector to obtain a recombinant plasmid I;

4) connecting the PRPP synthetase gene prs obtained in the step 2) to a recombinant plasmid I to obtain a recombinant plasmid II;

5. The method of claim 4, comprising the steps of:

1) knocking out a transcription repressor gene purR, a phosphogluconate dehydratase gene edd, an adenylate succinate synthetase gene purA and an inosinate dehydrogenase gene guaB of an escherichia coli W3110 genome respectively by using a P1 phage transduction method to obtain a mutant strain W3110 delta purR delta edd delta purA delta guaB;

3) Connecting the K326Q and P410W double mutation PRPP transamidase gene purF obtained in the step 2) to a plasmid vector pACYCDuet-1 to obtain a recombinant plasmid pACYCDuet 1-purF;

5) connecting PRPP synthetase gene prs obtained in the step 4) to the recombinant plasmid pACYCDuet1-purF obtained in the step 3) to obtain a recombinant plasmid pACYCDuet1-prs (D128A) -purF (K326Q, P410W);

6) introducing the recombinant plasmid pACYCDuet1-prs (D128A) -purF (K326Q, P410W) obtained in the step 5) into the receptor cell W3110 delta purR delta edd delta purA delta guaB obtained in the step 1) to obtain a recombinant bacterium.

6. Use of the recombinant bacterium of any one of claims 1 to 3 for the production of hypoxanthine and for the production of hypoxanthine derivatives.

7. The use of claim 6, wherein the recombinant bacteria are activated, and the activated recombinant bacteria are inoculated into LB liquid culture medium containing chloramphenicol for fermentation culture.

8. The application of claim 7, wherein the fermentation culture is completed after culturing the recombinant strain to OD600 of 8-12 under the conditions of culture temperature of 37 ℃, stirring speed of 400-800rpm and pH value of 6.5-7.5, adding IPTG (isopropyl thiogalactoside) as an inducer to a final concentration of 100 μ M and continuing feeding fermentation for 96h by using 50-80% by mass of glucose stock solution.

9. The use according to claim 7, wherein the concentration of chloramphenicol in LB liquid medium is 50 mg/L.

10. The use of claim 7, wherein the inoculation is to inoculate the recombinant strain seed liquid into LB liquid culture medium containing chloramphenicol according to the inoculation amount of 2-5% (v/v).